This proposal is to test the hypothesis that NHE3, the epithelial brush border Na/H antiporter, undergoes acute regulation by acting as a scaffold for some of its regulatory proteins and that the assembled regulatory complexes form in highly organized domains of the NHE3 C-terminus. The long-term goal is to understand how intestinal Na absorption is regulated as necessary background for developing drug therapy for diarrheal diseases based on the goal of pharmacologically stimulating NHE3 activity. The hypothesis underlying this grant will be tested by study of NHE3 regulation which centers on its C-terminal domain between aa 475 and 585, which is close to the N-terminal transport domain, and emphasizes the role of direct ezrin binding to NHE3, which was identified during the previous grant period as being necessary for multiple aspects of trafficking of NHE3. The Specific Aims of this grant are: Aim I: To understand how the C-terminal domain to which ezrin directly binds is involved in regulation NHE3 activity. We propose to define the role of direct ezrin binding to NHE3 on acute stimulation and inhibition of NHE3. We have identified that the domain of NHE3 which directly binds ezrin also interacts with multiple components of the PI-3K system and we will use mutagenesis to identify the role of each component on NHE3 activity. We will test the hypothesis that NHE3 trafficking is dependent on binding to the motor protein myosin VI. Aim II: We have identified that the phosphoinositides PIP2 and PIP3 appear to directly bind to NHE3 between aa 475 and 585. We will use mutagenesis and liposomal pull down assays to identify the domains of the NHE3 C-terminus through which interactions with the phosphoinositides occur and determine their role in NHE3 trafficking and basal and regulated activity. Studies will be carried out mostly in the polarized intestinal Na absorptive cell line, Caco-2 cells using mutagenesis, shRNA and dominant-negative constructs transiently infected via viral vectors, supplemented with commercially available knockout mice models of some of the NHE3 associating proteins with consequences determined using two-photon microscopy/SNARF-4F measurement of NHE3 activity in intact intestine.